Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors.

ABSTRACT

To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the Tm value. Rank ordering the affinity and predictions of the dissociation constant of compounds is obtained by measuring the increase in Tm for different protein-inhibitor complexes. This is the first time an extensive characterization of the TdCD method has been described for characterizing lead inhibitors in a drug discovery mode. The method has several favorable properties. Using the new six-cell Peltier temperature controller for the Jasco 810 spectropolarimeter, one can determine the affinity of 12-18 compounds per day. The method also requires only 20-40 microg protein per sample and can be used to estimate the affinity of compounds with dissociation constants of picomolar to micromolar. An important property of the method for lead discovery is that dissociation constants of approximately 5 microM can be estimated from a single experiment using a low concentration of compound such as 20 microM, which is generally low enough for most small molecules to be soluble for testing. In addition, the method does not require labeling the compound or protein. Although other methods such as isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of binding, ITC requires 1-2 mg protein per sample, cannot readily determine binding constants below nanomolar values, is most versatile with soluble compounds, and has a throughput of two to three experiments per day. The ITC method is not usually used in a high-throughput drug discovery mode; however, using the thermodynamic information from several ITC experiments can make the TdCD method very robust in determining reliable binding constants. Using the kinase inhibitors BMS-250595, purvalanol B, AG-12275, flavopiridol, and several other compounds, it is demonstrated that one can obtain excellent comparisons between the Kd values of binding to CDK2 obtained by TdCD and ITC.